Genome-Edited Maize Expressing Two Native Genes Confers Broad-Spectrum Resistance to Northern Corn Leaf Blight

Northern corn leaf blight (NCLB) can result in yield losses of up to 50% in maize. The most effective strategy for managing NCLB is the deployment of resistant hybrids. Conventional breeding methods typically require 6 or 7 backcross generations to introgress a resistance locus, often bringing along undesirable traits that reduce yield. Recent advances in genome editing offer a precise alternative, enabling the targeted incorporation of resistance genes without linkage drag. In this study, we identified an NCLB resistance gene, NLB18-R, that is allelic to Htn1 and Ht2/Ht3. Using CRISPR-Cas9, we replaced the susceptible allele (NLB18-S) with NLB18-R in an elite inbred, resulting in enhanced resistance to NCLB. In a parallel experiment, we inserted both NLB18-R and the resistance gene Ht1-R into preselected, closely linked sites on chromosome 1. Through genetic crossing, we combined these edits into a stack. The resulting genome-edited plants exhibited resistance to Setosphaeria turcica races 0, 1 and 23N. Field trials under disease-free conditions showed no significant yield differences between hybrids carrying NLB18-R, Ht1-R, or the stack compared to null and wild-type controls. These findings demonstrate that CRISPR-Cas9-mediated genome editing is a powerful tool for rapidly developing commercial-grade maize hybrids with broad-spectrum resistance to NCLB, and potentially other diseases.